Microtiter plates or microplates have become a standard tool in analytical research and clinical diagnostic testing laboratories. They are commonly used in a variety of detection procedures, which involve an image acquisition system such as fluorescence, luminescence, radiometric, absorbance, and light imaging. A microplate typically has 6, 24, 96, 384 or more sample wells arranged in a rectangular matrix and each well is designed to hold samples such as cell and tissue cultures as well as whole organisms such as the zebrafish.
In high-throughput microscopy, an image acquisition system, attached to a microscope, can be configured to image an entire well plate mounted onto the stage of the microscope, as a single image in one or more field of views, or each test well separately, also with one or more field of views. While most microplates consists of circular wells, rectangular wells are not uncommon. Each well of a microplate may hold between tens of nanoliters to several milliliters of liquid.
In some systems, a scan plan may be used to direct the imaging across the surface of the microplate. In the simplest method, the system must look at the entire microplate and identify the location of the test samples, which typically are cell and tissue cultures, or organisms. The test sample is then converted to a region envelope. The coordinates of the region are then mapped in the position space of the microscope stage. This allows the microscope motion to be programmed to cover the appropriate areas of the microplate, and avoid areas of waste between the tissue samples, or in the case of a microplate, sample wells that do not contain a sample. The scan plan may also be referred to as an illumination mask wherein, certain regions of the microplate that are masked out are not imaged. However, manually generating a scan plan can be time consuming and often may not be accurate.
Therefore an alternative process to provide high throughput image processing of microplates is desirable, such as a process to efficiently detects the microplate's sample well wall boundaries. Accurately identifying the location of the well wall can be used to speed up high-resolution image acquisition of relevant parts of the plate and may also be used in a pre-processing step for subsequent image acquisition and image analysis of the microplate. As such, a mask of the inner well regions can help to avoid unnecessary processing outside the well wall and improve the speed of downstream image processing. A mask can also help to reduce the complexity of illumination correction or flat field correction where there are bright spots within or outside the well.